The study was conducted to determine the effect of whole cottonseed (WCS), heat-treated whole cottonseed (HWCS), and whole cottonseed plus lanolin (WCS + Q on feed intake, milk production and milk composition. Eight Holstein heifers in early lactation were fed total mixed diets containing 0% WCS (basal), 25% WCS (WCS), 25% heat-treated WCS (HWCS), or 25% WCS with 2% lanolin (WCS + L). The eight heifers were divided into two groups of four and were fed the four diets according to the prescription of a switch-over design. Periods consisted of a 21-day adaptation period followed by a 16-day experimental period after which cows were switched to the next treatment. Milk samples for analysis were collected every fourth day during the 16-day experimental period. Dry matter intakes did not differ significantly between treatments although cows which received the HWCS diets had the highest intake. Milk yield was increased significantly (P < 0.05) on the WCS and HWCS treatments with the highest milk production on the HWCS treatment. Milk fat percentage and yield were increased (P < 0.01) on the WCS, HWCS and WCS+L treatments. Milk fat yield on the HWCS treatment was also higher (P < 0.05) than on the WCS and WCS+L treatments. Milk protein content or yield was not affected by any of the treatments. Milk fatty acid composition was altered significantly by all cottonseed treatments. WCS increased (P < 0.01) the weight of C18 and C18:1 fatty acids. The higher (P < 0.01) C18:2 content of milk fat on the HWCS treatment indicated that heat treatment protected fat from hydrogenation in the rumen. The inclusion of whole cottonseed or heat-treated whole cottonseed in total mixed diets for lactating dairy cows should lead to higher milk and milk fat production.